Folding of a large ribozyme during transcription and the effect of the elongation factor NusA

Abstract

We compared in vitro transcription-initiated folding of the ribozyme from Bacillus subtilis RNase P to refolding from the full-length, denatured state by monitoring the appearance of its catalytic activity. At 37 degrees C, Mg(2+)-initiated refolding of the wild type and a circularly permutate ribozyme takes minutes and is limited by a kinetic trap. Transcription by T7 RNA polymerase alters the folding pathway of both RNAs and introduces new kinetic traps. Transcription by the core Escherichia coli RNA polymerase yields the same result, in spite of its 4-fold-slower elongation rate. However, the presence of its elongation factor NusA accelerates more than 10-fold the transcription-initiated folding of the circularly, permutated ribozyme by E. coli RNA polymerase. The effect of NusA likely is caused by its enhancement of transcriptional pausing because NusA did not accelerate transcription-initiated folding using a mutant RNA polymerase that failed to pause or respond to NusA during ribozyme synthesis. We conclude that both transcription and specific pausing therein can alter RNA-folding pathways.

Figure 1

(A) Schematic representation of the domain structure of wild-type P RNA and CP RNA ribozymes and requirements for different substrates cleavage: processing of the pre-tRNA substrate requires folding of both the catalytic (C) and the specificity (S) domains, whereas proper folding of the C domain is sufficient for the cleavage of the selected substrate. The C domain in the P RNA is composed of a 5′ and a 3′ part interrupted by the S domain. (B) Refolding pathway for P RNA and CP RNA (5). Completely folded domains are indicated with the shaded boxes as in A; open symbols represent not-yet-folded domains. Essentially all wild-type P RNA fold through the slow, top pathway with a kinetic intermediate in which neither domain is folded. The CP RNA can fold through upper and lower pathways. The bottom pathway has an intermediate in which only the C domain is folded. (C) Cotranscriptional folding pathway. The initial intermediate formed during transcription differs from Mg²⁺-initiated refolding and probably has two new misfolds. (D) NusA effect on folding during transcription by E. coli RNAP. Here, folding of the S domain does not limit formation of native CP RNA. All folding rates are in s⁻¹ at 37°C.

Figure 2

Folding of P RNA and CP RNA during transcription by T7 RNAP. (A) The amount of P RNA synthesized [▴, S(t)] was measured by incorporation of [α-³²P]CTP. The amount of active P RNA was detected by catalytic activity with a tRNA substrate [A(t), ▵] or the selected substrate [A(t), ⋄]. The synthesis of P RNA has a transcription rate (k_s) of 1.3 ± 0.2 μM/min and an association constant (K) of 0.9 ± 0.2 μM⁻¹ according to Eq. 2. (B) Catalytically active fraction [A(t)/S(t)] for P RNA folding. The folding rate determined by cleaving the selected substrate is ≈4-fold faster than that from cleaving a tRNA substrate. (C) Catalytically active fraction for CP RNA folding.

Figure 3

(A) Synthesis of CP RNA by wild-type and mutant E. coli RNAP in the absence or in the presence of 90 nM NusA. The darkness of the bands reflects the dwell time at specific pausing sites. Asterisk indicates the pausing site at nucleotide U55 (225th position in the CP RNA). (B) Elongation rates of wild-type (solid bars) and mutant (shaded bars) E. coli RNAP were determined from single-round transcription assays on CP RNA template at the concentrations of NTP and NusA indicated below the bar graph. (C) Folding of CP RNA during transcription by 0.2 μM wild-type (Left) and mutant (Right) RNAP in the absence (●) and presence (□) of 0.4 μM NusA or by 0.2 μM wild-type enzyme at 0.06 mM (▵), 0.25 mM (♦), and 1 mM (curve fit) NTP. The synthesis of CP RNA by the E. coli RNAP is nonlinear and is best fit by a transcription rate (k_s) of 0.046 ± 0.016 μM/min and an association constant (K) of (0.20 ± 0.08) × 10⁻³ μM⁻¹ according to Eq. 2. The folding rates are determined by the cleavage of a pre-tRNA substrate.

Figure 4

Folding of CP RNA during transcription by 0.2 μM E. coli RNAP in the presence of 0.4 μM NusA assayed by cleavage of the selected substrate.